Journal: Scientific Reports

Article Title: Exclusive expression of KANK4 promotes myofibroblast mobility in keloid tissues

doi: 10.1038/s41598-024-59293-z

Figure Lengend Snippet: 3′ RNA-seq analysis identified keloid specific differentially expressed genes. ( a ) Scheme of dissections of the TAGLN-positive area, and workflow of the analysis performed in this study. ( b ) 3′ RNA-seq analysis for immature scars (n = 2) and keloids (n = 3). Volcano plots showing the differentially 112 upregulated genes and 108 downregulated genes in keloids (fold change > 2, FDR P -value ≤ 0.1). ( c ) Venn diagram showing the eight shared differentially expressed genes among the aforementioned 220 genes identified though the current 3′ RNA-seq analysis and 414 genes detected in the GSE113619 dataset (fold change > 2, FDR P -value ≤ 0.1). ( d ) Relative mRNA expressions of the eight differentially expressed genes in immature scars (n = 10) and keloids (n = 12) compared with GAPDH. Error bars show the mean ± SD. P -value was determined using the two-tailed t -test. * P < 0.05, ** P < 0.01, *** P < 0.001. ELN elastin, FDR false discovery rate, GAPDH glyceraldehyde-3-phosphate dehydrogenase, KANK4 KN motif and ankyrin repeat domains 4, KRT6A keratin 6A, OPCML opioid binding protein/cell adhesion molecule like, PTPRD protein tyrosine phosphatase receptor type D, qPCR quantitative polymerase chain reaction, RNA-seq RNA sequencing, S100A7 S100 calcium binding protein A7, TNXB tenascin XB, S100A8 S100 calcium binding protein A8, SD standard deviation, TAGLN transgelin.

Article Snippet: After blocking in 3% H 2 O 2 for 20 min, specimens were incubated with the primary antibodies for overnight: transgelin (TAGLN; ab14106, 1:200; abcam, Cambridge, UK), α-smooth muscle actin (α-SMA; M0858, 1:100; Dako, Glostrup, Denmark), KN motif and ankyrin repeat domains 4 (KANK4; ab121410, 1:50; abcam), opioid binding protein/cell adhesion molecule like (OPCML; ab238143, 1:1,000; abcam), S100 calcium binding protein A7 (S100A7; #45298, 1:400; Cell Signaling Technology, Danvers, MA, USA).

Techniques: RNA Sequencing, Two Tailed Test, Binding Assay, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: S100A7 correlates with adverse clinico-pathological features in ER-positive breast cancer patients. ( A ) Box plot depicting the differential expression of S100A7 in ER-positive and ER-negative BC patients as found in the METABRIC cohort; (****) p < 0.0001. ( B ) S100A7 expression correlates with a worse overall survival of ER-positive BC patients in the METABRIC cohort. ( C ) The expression levels of S100A7 were higher in ER-positive BC patients with more advanced tumor grade. (****) p < 0.0001; the number of patients of each group is indicated in the figure.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Quantitative Proteomics, Expressing

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: IGF-1 induces the expression of S100A7. ( A ) mRNA expression of S100A7 in MCF-7 and T47D cells treated vehicle or IGF-1 (10 nM, 48 h), as evaluated by qRT-PCR. Values were normalized to the 36B4 gene expression and shown as fold changes of mRNA expression induced by IGF-1 compared to cells treated with vehicle. Data are mean ± SEM of at least three independent experiments performed in duplicate. ( B ) Transactivation of a S100A7 (pS100A7) promoter construct observed in MCF-7 and T47D cells treated with IGF-1 (10 nM, 18 h). Luciferase activity was normalized to the internal transfection control. Results are expressed as the % change of normalized RLU values relative to vehicle-treated cells. Data shown are the mean ± SEM of at least two independent experiments performed in triplicate. ( C , D ) Representative immunoblots showing the increase of S100A7 protein expression in MCF-7 and T47D cells treated with IGF-1 (10 nM, 48 h). β-actin serves as loading control. Lower panels show densitometric analysis of the blots normalized to β-actin. Data shown are the mean ± SEM of at least two independent experiments. (*) p < 0.05; (**) p < 0.01.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Construct, Luciferase, Activity Assay, Transfection, Control, Western Blot

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: IGF-1R mediates the up-regulation of S100A7 expression induced by IGF-1. The upregulation of S100A7 protein expression observed in MCF-7 ( A ) and T47D ( C ) cells treated with vehicle (–) or IGF-1 (10 nM IGF-1, 48 h) was abrogated by silencing IGF-1R, as evidenced by western blotting. Representative immunoblots showing the efficiency of IGF-1R silencing in MCF-7 ( B ) and T47D cells ( D ). The up-regulation of S100A7 protein levels induced by IGF-1 (10 nM, 48 h) was prevented in MCF-7 cells knock out for IGF-1R (MCF-7 KO-IGF1R) compared to MCF-7 control cells (MCF-7 cas9), as indicated ( E ). Representative immunoblot showing the efficiency of IGF-1R knock-out ( F ). β-actin serves as loading control. Side panels show densitometric analysis of the blots normalized to β-actin. Data shown are the mean ± SEM of at least two independent experiments. (*) p < 0.05; (***) p < 0.001.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Expressing, Western Blot, Knock-Out, Control

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: ERK1/2, AKT, and STAT3 signaling pathways are involved in the up-regulation of S100A7 expression induced by IGF-1. Phosphorylation of IGF-1R ( A ), ERK1/2 ( B ), and AKT ( C ) in MCF-7 cells treated with IGF-1 (10 nM, 24 h), alone and in combination with the IGF-1R inhibitor OSI-906 (OSI, 10 μM) ( A ), the MEK inhibitor PD0325901 (PD, 300 nM) ( B ), as well as the PI3K/AKT inhibitor LY294002 (LY, 10 μM) ( C ). In MCF-7 ( D ) and T47D ( E ) cells, the exposure to IGF-1 (10 nM, 24 h) induced the phosphorylation of STAT3 (Y705), which was prevented in the presence of the JAK/STAT inhibitor Tyrphostin AG490 (AG, 25 μM). ( F ) STAT3 phosphorylation (Y705) in response to IGF-1 (10 nM, 24 h) is absent in MCF-7 cells knock-out for IGF-1R (MCF-7 KO-IGF1R) via CRISPR genome editing, but present in MCF-7 control cells (MCF-7 cas9). The up-regulation of S100A7 protein expression was prevented in MCF-7 ( G ) and T47D ( H ) cells treated with IGF-1 (10 nM, 48 h), alone and in combination with OSI (OSI-906, 10 μM), AG (AG490, 25 μM), PD (PD0325901, 300 nM), and LY294002 (LY, 10 μM). β-actin, IGF-1R, ERK1/2, AKT, STAT3 serve as loading controls, as indicated. Data shown are the mean ± SEM of at least two independent experiments. Evaluation of luciferase activity in MCF-7 ( I ) and T47D ( J ) cells transiently transfected with a S100A7 (pS100A7) promoter construct and treated with IGF-1 (10 nM, 18 h) in the presence of OSI (10 μM), AG (25 μM), PD (300 nM), and LY (10 μM). Luciferase activity was normalized to the internal transfection control. Results are expressed as the % change of normalized RLU values relative to vehicle-treated cells. Data shown are the mean ± SEM of at least two independent experiments performed in triplicate. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Protein-Protein interactions, Expressing, Phospho-proteomics, Knock-Out, CRISPR, Control, Luciferase, Activity Assay, Transfection, Construct

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: STAT3 is involved in the transcriptional activation of S100A7 by IGF-1. ( A ) IGF-1 (10 nM, 24 h) induces the recruitment of STAT3 (Y705) to the STAT3 binding site located within the S100A7 promoter sequence, as ascertained by Chromatin Immunoprecipitation (ChIP)-RT-PCR assay performed in MCF-7 cells. In control samples, nonspecific IgG was used instead of the primary antibody. Data shown are the mean ± SEM of at least two independent experiments performed in duplicate. ( B ) Representative immunoblots of HA, pSTAT3 (Y705), and STAT3 in MCF-7 cells which were transiently transfected with pcDNA3 and STAT3-HA for 48 h, to generate a constitutively activated STAT3 system. ( C ) Evaluation of S100A7 mRNA expression by RT-PCR in MCF-7 cells transfected with pcDNA3 and STAT3-HA for 48 h, as indicated. 36B4 gene serves as normalizing control. ( D ) The transactivation of a S100A7 (pS100A7) promoter plasmid observed after treatment with IGF-1 (10 nM, 18 h) is enhanced in MCF-7 cells transfected for 48 h with STAT3-HA, compared with cells transfected with pcDNA3 control vector. Results are expressed as the % change of normalized RLU values relative to vehicle-treated cells. Data shown represent the mean ± SEM of at least two independent experiments performed in triplicate. (*), (#) p < 0.05; (**) p < 0.01; (***) p < 0.001. Side panel shows a schematic representation of human S100A7 promoter carrying the STAT3-responsive site.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Activation Assay, Binding Assay, Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: Paracrine release of S100A7 by BC cells induces proliferative effects in HUVECs. ( A ) Evaluation of S100A7 concentration in conditioned medium from MCF-7 cells treated with vehicle or IGF-1 (10 nM, 48 h), as evaluated by ELISA. Data shown are the mean ± SEM of at least three independent experiments performed in duplicate. ( B ) HUVECs were cultured in medium collected from MCF-7 cells treated with either vehicle or IGF-1 (10 nM, 48 h), in the presence of the RAGE inhibitor FPS-ZM1 (2 μM). Thereafter, HUVECs were subjected to SRB assay for the evaluation of cell growth. ( C ) HUVECs were cultured in 0.1% FBS in the presence of vehicle and human recombinant S100A7 (0.15μg/mL), alone and in combination with the RAGE inhibitor FPS-ZM1 (2 μM), as indicated. After 48 h, HUVECs were subjected to SRB assay for the evaluation of cell growth. Data shown are the mean ± SEM of at least 2 independent experiments performed in sextuplicate. (*) p < 0.05; (***) p < 0.001.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Sulforhodamine B Assay, Recombinant

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: IGF-1 triggers the formation of endothelial tubes by activating the S100A7/RAGE signaling. ( A ) HUVECs were transfected with siRAGE (10 nM) or non-targeting scramble siRNA for 24 h. Thereafter, tube formation assay was performed on HUVECs, which were detached and cultured in medium collected from MCF-7 cells treated with either vehicle or IGF-1 (10 nM, 48 h). Tube formation was evaluated 8 h after HUVEC seeding. ( B ) Efficacy of RAGE silencing in HUVECs. ( C – E ) Quantification of the number of tubes, number of nodes and number of master junctions observed in HUVECs, as indicated. Data shown are the mean ± SEM of at least two independent experiments performed in duplicate. (**) p < 0.01.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Transfection, Tube Formation Assay, Cell Culture

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: S100A7 triggers endothelial tube formation through the involvement of RAGE. ( A ) HUVECs were transfected with siRAGE (10 nM) or non-targeting scramble siRNA for 24 h. Thereafter, tube formation assay was performed on HUVECs, which were detached and cultured in Endothelial Basal Medium supplemented with either vehicle or S100A7 (0.15 μg/mL). Tube formation was evaluated 8 h after HUVEC seeding. ( B ) Efficacy of RAGE silencing in HUVECs. ( C – E ) Quantification of the number of tubes, number of nodes and number of master junctions observed in HUVECs, as indicated. Data shown are the mean ± SEM of at least two independent experiments performed in duplicate. (*) p < 0.05; (**) p < 0.01.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Transfection, Tube Formation Assay, Cell Culture

Journal: Cancers

Article Title: Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

doi: 10.3390/cancers13040621

Figure Lengend Snippet: S100A7/RAGE signaling prompts endothelial tube formation. ( A ) Tube formation assay was performed in HUVECs which were cultured in medium collected from MCF7-Ex Neg (control) and MCF7-S100A7 (overexpressing S100A7) cells, in the presence of the RAGE inhibitor FPS-ZM1 (2 μM). Tube formation was evaluated 8 h after HUVEC seeding. ( B , C ) Quantification of the number of tubes and number of nodes observed in HUVECs, as indicated. Data shown are the mean ± SEM of at least two independent experiments performed in duplicate. (*) p < 0.05; (***) p < 0.001.

Article Snippet: Cells were processed according to the previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% ( w / v ) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance).

Techniques: Tube Formation Assay, Cell Culture, Control

Journal: Endocrinology

Article Title: Myosin VIIa Supports Spermatid/Organelle Transport and Cell Adhesion During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2018-00855

Figure Lengend Snippet: Antibodies Used for Different Experiments in This Study

Article Snippet: Working Dilution IB Analysis IF Analysis Myosin VIIA (RRID: AB_2148626 ) ( 34 ) Mouse Santa Cruz Biotechnology sc-74516 1:200 1:100 Myosin VIIA (RRID: AB_303841 ) ( 35 ) Rabbit Abcam ab3481 1:5000 1:400 EB1 (RRID: AB_397891 ) ( 36 ) Mouse BD Biosciences 610534 — 1:200 ZO-1 (RRID: AB_2533938 ) ( 37 ) Rabbit Invitrogen 61-7300 1:250 1:100 Occludin (RRID: AB_2533977 ) ( 38 ) Rabbit Invitrogen 71-1500 1:250 1:100 CAR (RRID: AB_2087557 ) ( 39 ) Rabbit Santa Cruz Biotechnology sc-15405 1:200 1:50 JAM-A (RRID: AB_2533241 ) ( 40 ) Rabbit Invitrogen 36-1700 1:250 — Claudin 11 (RRID: AB_2533259 ) ( 41 ) Rabbit Invitrogen 36-4500 1:250 — N-cadherin (RRID: AB_2313779 ) ( 42 ) Mouse Invitrogen 33-3900 — 1:100 N-cadherin (RRID: AB_647794 ) ( 43 ) Rabbit Santa Cruz Biotechnology sc-7939 1:200 — β -Catenin (RRID: AB_2533982 ) ( 44 ) Rabbit Invitrogen 71-2700 1:250 1:100 Arp3 (RRID: AB_476749 ) ( 45 ) Mouse Sigma-Aldrich A5979 1:3000 1:50 Eps8 (RRID: AB_397544 ) ( 46 ) Mouse Invitrogen 610143 1:5000 1:50 N -WASP (RRID: AB_2288632 ) ( 47 ) Rabbit Santa Cruz Biotechnology sc-20770 1:200 — Plastin 3 (RRID: AB_2636816 ) ( 48 ) Rabbit Abcam ab137585 1:500 — β 1 -Integrin (RRID: AB_2130101 ) ( 49 ) Rabbit Santa Cruz Biotechnology sc-8978 — 1:50 α -Tubulin (RRID: AB_2241126 ) ( 50 ) Mouse Abcam ab7291 — 1:200 Detyrosinated α -tubulin (RRID: AB_869990 ) ( 51 ) Rabbit Abcam ab48389 — 1:200 β -Tubulin (RRID: AB_2210370 ) ( 52 ) Rabbit Abcam ab6046 1:5000 1:200 Tyr-tubulin (RRID: AB_305328 ) ( 53 ) Rat Abcam ab6160 — 1:200 Acetylated α -tubulin (RRID: AB_448182 ) ( 54 ) Mouse Abcam ab24610 — 1:200 β -Actin (RRID: AB_630836 ) ( 55 ) Goat Santa Cruz Biotechnology sc-1616 1:200 — Vimentin (RRID: AB_628437 ) ( 56 ) Mouse Santa Cruz Biotechnology sc-6260 1:200 1:200 Gapdh (RRID: AB_2107448 ) ( 57 ) Mouse Abcam ab8245 1:1000 — Laminin γ 3 (RRID: AB_2636817 ) ( 58 ) Rabbit Cheng Laboratory ( 67 ) — — 1:200 Goat IgG-HRP (RRID: AB_634811 ) ( 59 ) Bovine Santa Cruz Biotechnology sc-2350 1:3000 — Rabbit IgG-HRP (RRID: AB_634837 ) ( 60 ) Bovine Santa Cruz Biotechnology sc-2370 1:3000 — Mouse IgG-HRP (RRID: AB_634824 ) ( 61 ) Bovine Santa Cruz Biotechnology sc-2371 1:3000 — Rabbit IgG–Alexa Fluor 488 (RRID: AB_2576217 ) ( 62 ) Goat Invitrogen A-11034 — 1:250 Rabbit IgG–Alexa Fluor 555 (RRID: AB_2535850 ) ( 63 ) Goat Invitrogen A-21429 — 1:250 Mouse IgG–Alexa Fluor 488 (RRID: AB_2534088 ) ( 64 ) Goat Invitrogen A-11029 — 1:250 Mouse IgG–Alexa Fluor 555 (RRID: AB_141780 ) ( 65 ) Goat Invitrogen A-21424 — 1:250 Rat IgG–Alexa Fluor 488 (RRID: AB_141373 ) ( 66 ) Goat Invitrogen A-11006 — 1:250 Open in a separate window Antibodies used herein cross-reacted with the corresponding proteins in rats.

Techniques: